This invention relates to a method and apparatus for quantitative analysis with color identification test paper.
Qualitative or quantitative analyses, conducted by a "Dip-and-Read" process with color identification test paper (hereinafter referred to as "test paper"), is widely used for such applications as the measurement of chemical constituents in blood and the screening examination of urine, and others by virtue of simplicity and ease of handling. Such analysis is particularly useful where rapid measurements or large numbers of measurements are required. When a technician performs the analysis by sight, the tone of coloration of the test paper painted with or dipped in the liquid to be examined is visually compared with standard color samples. This is suited for conducting only qualitative analysis, or, at best, rough quantitative analysis. In order to enhance to accuracy of quantitative analysis, it is necessary to employ some analyzing device or reflectivity meter which is capable of photoelectrically measuring the tone of coloration of the test paper. In recent years, therefore, along with the development and improvement of new types of test papers, there have been provided analyzers of various types, enabling a user to execute simplified and expeditious techniques of quantitative analysis on many samples.
Prior art analyzers provided until now operate with an indirect measuring method which measures a change of the reflectivity of the test paper photoelectrically, produces electric signals, and then converts the signals into differences of concentration by the application of a calibration curve. However, prior art analyzers are insufficiently accurate. Consequently, such analysis using test paper is said to be less accurate than titration and other methods of quantitative analysis, which latter methods are neither as simple nor as easy.
The principle of operation of these analyzers is based upon the fact that the reflection spectrum of the colored test paper should vary depending on the concentrations Y.sub.1, Y.sub.2, Y.sub.3 of the target substance in the liquid to be examined, as shown in FIG. 1, wherein light with an appropriate wavelength, in particular with a wavelength or its adjacent one (.lambda.0 in FIG. 1), whose reflectivity changes markedly in accordance with the changes of the concentration, is selected when the change of the reflectivity of the test paper at the same wavelength is measured photoelectrically and indicated, being converted into units of concentration by reference to a calibration curve of the reflectivity versus concentration such as is shown in FIG. 2.
Of these types of analyzers, one is heretofore in wide use which is equipped (in the section assigned to the measurement of reflectivity) with an integrating sphere capable of efficiently measuring the reflected ray from the surface of reflection. FIG. 3 is a diagram of an example of the analyzers which use such an integrating sphere. The light from the light source 2 mounted on the upper part of the integrating sphere 1 is filtered to a predetermined wavelength through a filter 3 and irradiates the test paper 5 set under a specimen window 4 at the bottom of the integrating sphere. From the surface of the test paper 5 is reflected a quantity of light corresponding to the degree of coloration of the test paper. This reflected light reflects diffusively in the interior of the integrating sphere 1 and irradiates an optical detector 6 provided on a side face of the integrating sphere.
The magnitude of the reflected light represented by the output signal from the optical detector 6 passes through an amplifying-measuring circuit 7 and is indicated on the meter 8, which has a scale for direct reading of the concentration. In order to measure reflectivity, a standard reference signal must be established. In so doing, a relative reflectivity is obtained by comparing the magnitude of the reference signal with the magnitude of the output signal produced by the reflected light from the test paper. The input signal to the meter 8 is based on this relative reflectivity. There are various ways of establishing the reference signal: providing an electrical reference signal in advance, providing a standard of known reflectivity to enable the quantity of reflected light from the standard to be stored as a reference signal during measurement, and so on.
This type of prior art device has the following disadvantages:
1. It is constructed so as to photoelectrically measure the changes in reflectivity. Consequently, it is difficult to prevent the occurrence of dark current in the optical detector 6 and the offset voltage caused by the amplifier in the amplifying-measuring circuit 7. Dark current or offset voltage of photoelectric detectors also drift due to changes in their working temperature.
This drift can be fully corrected if the light from the light source 1 is interrupted by the use of, for example, a chopper or an intermittent light producing circuit, which has been already adopted in some devices. The influence of stray rays introducing into the integrating sphere 1 from outside also can be eliminated by the same means. In these cases, the fluctuations of the above-mentioned drift or the stray rays from outside must be, needless to say, small enough in comparison with the intermittent cycle of the flux of light.
2. The interior of the integrating sphere 1 is easily contaminated, since fibrous flocks of the test paper, dust from outside, or liquid to be examined can stick thereto. That causes the diffusion of the light to vary, causing the characteristics of the integrating sphere 1 to change during the course of long-time use. To prevent this contamination, a transparent plate 9 (such as sheet glass) is fitted into the specimen window 4 in order to prevent the intrusion of dust and the like into the interior. However, the operator must wipe off dirt on the outer surface of the transparent plate 9 before the measurement operation.
Such a transparent plate 9 on the specimen window 4 causes a problem in that a portion of the light which is supposed to reach the test paper 5 is reflected by the transparent plate 9 itself, generating inner stray rays which reach the optical detector 6. Even assuming that an object of 0% reflectivity were measured in conformity with the characteristics of the integrating sphere 1, detector 6 would still have a non-zero output. Therefore, the calibration curve of the output from detector 16 as compared to actual reflectivity does not pass through the origin. These inner stray rays vary, depending on the inclination of the transparent plate 9 with respect to the optic axis of incidence and also depending on the scattering of the incident light, which indicator and scattering vary between different analyzers. Hence, where test papers are measured in a reflectivity measuring system which has a transparent plate 9, different analyzers can yield different results, even though test papers of the same reflectivity were measured. This is shown in FIG. 4, in which Ro is the actual reflectivity of a standard of known reflectivity and the ordinate is relative reflectivity when corrected to Ro=100. The calibration curve passes through the origin in the case of a device M1 which has no inner stray rays, while the calibration curve deviates from the origin in devices M2 and M3 which have increasing amounts of stray rays.
There are other errors originating from other rays reflected by the inner parts of the optical system including the integrating sphere, such as the peripheral region of the specimen window 4, the shield plate, from the light from the light source and so forth. The inner stray rays caused by the latter can be excluded to some extent by changing the shape and structure of the optical system, e.g. by redesigning the integrating sphere. Still, some of these rays remain as an instrumental error, similar to the case of the inner stray rays discussed above. Consequently, the calibration curve is inaccurate. This brings about instrumental error, obstructing accurate analysis using test paper.
The conversion of relative reflectivity thus obtained into a directly readable concentration value is generally carried out by equipping meter 8 with a scale plate 10 having a nonlinear direct-reading scale, in analog form, this being calibrated into concentration, as is shown in FIG. 3. There is also another method, in which the measured output signal is corrected when it is compared with the reference signal so that both signals will be identical to each other. Here, the correction is determined by rotation of a potentiometer, and the concentration is read with the help of a scale plate attached to the potentiometer.
In either case mentioned above, however, the scale plates are paired with corresponding types of test papers to be examined. Accordingly, if the inner stray rays and the offset voltages differ between measuring devices, different kinds of scale plates should be provided on individual devices. This is impractical. Usually, devices share only one kind of scale plate having an engraved scale relative to one item to be examined. It is, thus, unavoidable that analyzers exist which have varying instrumental errors.
3. Even if an analyzer without any instrumental error could be made, it is impossible to fabricate test paper having a reflecting spectrum which is flat to all wavelengths of incident light. It is also inevitable that the wavelength of the light source will shift to some extent. If the wavelength of the light source shifts, the calibration curve changes, since the reflectivity of the test paper varies depending on wavelength, thereby resulting in other instrumental errors.
FIGS. 5 and 6 illustrate these problems. FIG. 5 illustrates the correlation between the reflecting spectra of the test papers at concentrations Y1, Y2, and Y3 at two different wavelengths (.lambda.0 and .lambda.1) of incident light thereby showing that the reflectivity r shifts with wavelength. FIG. 6 illustrates the different calibration curves of concentration with respect to reflectivity at the two different wavelengths .lambda.0 and .lambda.1 shown in FIG. 5.
The sources of error mentioned above vary from analyzer to analyzer at the time of manufacture. But even in the same analyzer, the intensity of light from the light source varies, and the wavelength fluctuates as the conditions of supply voltage, ambient temperature, and other parameters change both at the time of manufacture and later during use. This means that the characteristics of the analyzer itself change with the passage of time.